1.Tazobactam inactivation of SHV-1 and the inhibitor-resistant Ser130 -->Gly SHV-1 beta-lactamase: insights into the mechanism of inhibition.
Pagan-Rodriguez D1, Zhou X, Simmons R, Bethel CR, Hujer AM, Helfand MS, Jin Z, Guo B, Anderson VE, Ng LM, Bonomo RA. J Biol Chem. 2004 May 7;279(19):19494-501. Epub 2004 Feb 2.
The increasing number of bacteria resistant to combinations of beta-lactam and beta-lactamase inhibitors is creating great difficulties in the treatment of serious hospital-acquired infections. Understanding the mechanisms and structural basis for the inactivation of these inhibitor-resistant beta-lactamases provides a rationale for the design of novel compounds. In the present work, SHV-1 and the Ser(130) --> Gly inhibitor-resistant variant of SHV-1 beta-lactamase were inactivated with tazobactam, a potent class A beta-lactamase inhibitor. Apoenzymes and inhibited beta-lactamases were analyzed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), digested with trypsin, and the products resolved using LC-ESI/MS and matrix-assisted laser desorption ionization-time of flight mass spectrometry. The mass increases observed for SHV-1 and Ser(130) --> Gly (+ Delta 88 Da and + Delta 70 Da, respectively) suggest that fragmentation of tazobactam readily occurs in the inhibitor-resistant variant to yield an inactive beta-lactamase.
2.Characterization of recombinant human coagulation factor XFriuli.
Kim DJ1, Girolami A, James HL. Thromb Haemost. 1996 Feb;75(2):313-7.
Naturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational gamma-carboxy-glutamic acid (Gla) and beta-hydroxy aspartic acid (beta-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels.
3.The tandem chain extension aldol reaction used for synthesis of ketomethylene tripeptidomimetics targeting hPEPT1.
Thorn K1, Nielsen CU, Jakobsen P, Steffansen B, Zercher CK, Begtrup M. Bioorg Med Chem Lett. 2011 Aug 1;21(15):4597-601. doi: 10.1016/j.bmcl.2011.05.108. Epub 2011 Jun 6.
The rationale for targeting the human di-/tripeptide transporter hPEPT1 for oral drug delivery has been well established by several drug and prodrug cases. The aim of this study was to synthesize novel ketomethylene modified tripeptidomimetics and to investigate their binding affinity for hPEPT1. Three related tripeptidomimetics of the structure H-Phe-ψ[COCH(2)]-Ser(Bz)-X(aa)-OH were synthesized applying the tandem chain extension aldol reaction, where amino acid derived β-keto imides were stereoselectively converted to α-substituted γ-keto imides. In addition, three corresponding tripeptides, composed of amide bonds, were synthesized for comparison of binding affinities. The six investigated compounds were all defined as high affinity ligands (K(i)-values <0.5 mM) for hPEPT1 by measuring the concentration dependent inhibition of apical [(14)C]Gly-Sar uptake in Caco-2 cells. Consequently, the ketomethylene replacement for the natural amide bond and α-side chain modifications appears to offer a promising strategy to modify tripeptidic structures while maintaining a high affinity for hPEPT1.
4.A convenient incorporation of conformationally constrained 5,5-dimethylproline into the ribonuclease A 89-124 sequence by condensation of synthetic peptide fragments.
Cerovský V1, Welker E, Scheraga HA. J Pept Res. 2003 Mar;61(3):140-51.
The presence of l-5,5-dimethylproline (dmP) within an amino acid sequence results in the formation of an X-dmP peptide bond predominantly locked in a cis conformation. However, the common use of this unnatural amino acid has been hampered by the difficulty of the economical incorporation of the dmP residue into longer peptide segments due to the steric hindrance imposed by the dimethyl moieties. Here, we describe synthesis of the C-terminal 36-residue peptide, corresponding to the 89-124 sequence of bovine pancreatic ribonuclease A (RNase A), in which dmP is incorporated as a substitute for Pro93. The peptide was assembled by condensation of protected 5- and 31-residue peptide fragments, which were synthesized by solid-phase peptide methodology using fluorenylmethyloxycarbonyl chemistry. We focused on optimizing the synthesis of the Fmoc-Ser(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-dmP-OH pentapeptide (residues 89-93) with efficient acylation of the sterically hindered dmP residue.